Polysome profile analysis is a frequently performed task in translational control research that not only enables direct monitoring of the efficiency of translation but can easily be extended with a wide range of downstream applications such as Northern and western blotting, genome-wide microarray analysis or qRT-PCR. Here, we describe a method for the isolation and quantification of high-quality polysome-bound mRNA complexes from small quantities of liquid-nitrogen-frozen solid tissue samples of rice shoots/roots. The mRNA obtained can be further analyzed by methods that evaluate polysomal mRNA abundance at the individual transcript or global level.

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David SeccoAffiliation 1: Department of Plant Molecular Biology, University of Lausanne, Lausanne, SwitzerlandAffiliation 2: Australian Research Council Centre of Excellence in Plant Energy Biology, The University of Western Australia, Perth, AustraliaBio-protocol author page:a2029

Yves PoirierAffiliation: Australian Research Council Centre of Excellence in Plant Energy Biology, University of Western, Ontario, CanadaBio-protocol author page:a2033

[Abstract]
Polysome profile analysis is a frequently performed task in translational control research that not only enables direct monitoring of the efficiency of translation but can easily be extended with a wide range of downstream applications such as Northern and western blotting, genome-wide microarray analysis or qRT-PCR. Here, we describe a method for the isolation and quantification of high-quality polysome-bound mRNA complexes from small quantities of liquid-nitrogen-frozen solid tissue samples of rice shoots/roots. The mRNA obtained can be further analyzed by methods that evaluate polysomal mRNA abundance at the individual transcript or global level.

For one gradient,
carefully add 1.85 ml of the 50% sucrose solution, 3.65 ml of the 35%,
3.65 ml of the 20% (w/v) and again 1.35 ml of 20% (w/v) to Beckman tubes
(12 ml total volume). Prepare gradients at least one day before cell
lysis to allow gradient to diffuse overnight at 4 °C.

Cover the
tube with parafilm and incubate at 4 °C overnight. Alternatively,
sucrose gradients can be stored at -80 °C indefinitely.

30-day-old rice roots or shoots were harvested, frozen and ground to powder in liquid nitrogen.

150 mg of powder was combined with 1.2 ml of chilled polysome buffer.

Debris was removed by centrifugation at 16,000 x g for 15 min at 4 °C.

Aliquots of the resulting supernatant were transferred (about 500
μl) to a new pre-chilled 1.5 ml tube. Measure OD260nm for each sample
using NanoDrop.

Load the same OD amount of lysate onto each
gradient on to 20 to 50% (w/w) continuous sucrose gradients. Keep 10% of
lysates as an input to determine the cytosolic steady-state mRNA
levels.

Weight and balance each gradient before ultracentrifugation.

Centrifuge at 175,000 x g for 165 min at 4 °C using SW41Ti rotor in a Beckman Coulter.

While the samples are centrifuging, clean fraction collector with
warm MilliQ water containing a bit of RNase decontamination solution.

Carefully remove tubes from the rotor and place them at 4 °C until they are ready for running.

Switch on computer, pump, UV-Vis Spectrophotometer and fraction
collector. Set pump at 2.3 ml/min and the fraction collector by time.
Place 10 ml tubes on fraction collector.

Open Cary WinUV software.

Fractions were collected from the bottom to the top of the gradient
with continuous monitoring of the absorbance at 254 nm. Run chasing
solution [20% (w/v) sucrose] with bromophenol blue through the system
until it reaches the needle. Make sure to see at least one drop coming
out of the needle such that no bubbles are introduced into the gradient.

Begin running the chasing solution through the gradient. Run solution with the pump at 2.3 ml/min.

Click on acquire data button and press run on the fraction collector.

Place fractions on dry ice.

Polysomal RNA extraction

Add 4 ml of Trizol reagent to each tube

Isolate RNA according to manufacturer’s Trizol protocol.

Measure the RNA concentration of each fraction or input.

Representative data

We used a simple and highly reproducible method for the analysis of polysomes in rice (Oryza sativa). All the experiments we made, displayed a very similar profiles, however, some differences can be observed in the form of the profiles when the plants are stressed and also in the older plants. In the literature, analysis of polysome profiles issued from different plant species show a variability between species and plant organs as shown in Figure 2 below.

Note: We used a simple and highly reproducible method for the analysis of polysomes in rice (Oryza sativa). All the experiments we made, displayed a very similar profiles, however, some differences can be observed in the form of the profiles when the plants are stressed and also in the older plants. In the literature, analysis of polysome profiles issued from different plant species show a variability between species and plant organs (see examples in Figure 2).

Notes

To isolate the RNA form fractions from sucrose density centrifugation for polysome analysis, it’s important to keep in mind that TRizol purification could be the best option in case of large scale extraction (Rice produce more biological materiel than Arabidospis). In case of small samples (particularly Arabidopsis roots samples) the use of RNeasy kit purification of RNA will be more cost-effective but sometimes the quality of the RNA would be not of high quality for subsequent qPCR analysis. It’s important to centrifuge the Trizol extract at 12,000 x g for 15 min to separate the phases. We have to sacrifice a layer of the aqueous phase (containing RNA) at the interphase, so that we will carry over proteins and phenol to the RNA.

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